In multi-cellular organisms, cells must communicate with each other in order for growth and development to occur in an ordered manner. In animals, it has long been known that polypeptides act as signalling molecules in mediating communication between cells, a common example being insulin in humans. These signalling molecules are responsible for initiating many cellular processes, typically by binding to a receptor at the cell surface, which in turn transmits a message to inside the cell via downstream signalling proteins such as membrane associated protein kinases (MAPK), tyrosine phosphatases and Ras proteins. In the cell, the cell signalling pathway end-point is usually a transcription factor target, which mediates a change in gene expression in the cell, thus causing a change in the growth and/or development of the cell in response to the initial extracellular signal.
In plants, it is also known that cell signalling occurs, and this was thought to be mediated by plant hormones such as auxin and cytokinin. More recently, the discovery of systemin has shown that polypeptides also play a role in cell-signalling in plants. One of the largest families of signalling polypeptides identified in plants is the Clavata3 (clv3)/Endosperm Surrounding Region (ESR)-related (CLE) family. These proteins are the most highly characterised family of small polypeptides in plants. The Arabidopsis thaliana genome contains 32 CLE genes. Clv3 is the best characterised CLE family member which acts together with a receptor kinase (CLAVATA 1) to play a role in regulating the proliferation of cells in the shoot (apical) meristem. At present, however, most of the CLE family remain functionally undefined.
The CLE gene family has been shown to be present in a variety of other plant species (Jun et al Cell. Mol. Life. Sci. 65 743-755 (2008) and Frickey et al BMC Plant Biology 2008, 8:1 10.1186/1471-2229-8-1) including rice, maize, tomato and alfalfa.
The polypeptides encoded by the CLE genes share common characteristics. They are less than 15 kDa in mass and comprise a short stretch of hydrophobic amino acids at the amino terminus which serves to target the polypeptide to the secretory pathway. This conserved stretch of 14 amino acids is known as the CLE domain (Jun et al supra).
Higher plants show post-embyronic development at shoot and root tips, which are known as the apical meristems. Stem cells at these meristems produce cells which differentiate to become flower, leaf, stem or root cells. A loss-of-function mutant resulting in an excess of stem cells at the apical meristem suggests that Clv3 plays a role in regulation of growth and/or differentiation at the growing tip. Over expression of CLV3 results in loss of apical stem cells, thus post-embryonic above ground parts of the plant are lost. The signalling pathway which CLV3 regulates has been elucidated and is described in Jun et al (supra). This pathway is thought to be conserved amongst other plants species.
Shiu and Bleecker suggest that the CLE family is likely to coordinate with a group of plant receptors known as the leucine-rich-repeat receptor-like (LLR-RLK) kinases (PNAS 98 10763-10768 (2001)).
U.S. Pat. No. 7,179,963 describes a maize clv3-like nucleotide sequence, and its use in modulating plant development and differentiation. U.S. Pat. No. 7,335,760 discloses nucleic acid sequences for use in genetically modifying a plant to increase plant yield and the mass of the plant, for example for biofuel production.
Other CLE family members have been shown to inhibit cell differentiation. For example, Frickey et al (supra) have looked at the CLE family and suggested that CLE family members CLE41 and/or 42 may play a role in vascular development. Ito et al (Science Vol 313 842-845 (2006)) show that dodecapeptides are important in preventing vascular cell differentiation.
In contrast, however, Strabala et al (Plant Physiology vol. 140 1331-1344 (2006)) show that CLE41 and/or 42 are genuine expressed members of the CLE family. Although general over-expression of CLE42 throughout the plant results in a dwarf phenotype, Strabala et al report that CLE42 is likely to be a functionally redundant molecule.
The source of biomass in plants is their woody tissue, derived from the vascular meristems of the plant such as the cambium and procambium, which divide to form the phloem and xylem cells of the vascular tissue within the plant stems and roots. The cambium and procambium (collectively known as the vascular meristems) are growth zones which enable the plant to grow laterally, thus generating the majority of biomass. Enhancing lateral growth by genetically altering the rates of procambial or cambial cell division may lead to an increase in the plant biomass. This would provide an additional source of biomass for various industries dependent upon plant derived products, such as the biofuel or paper industries.
Increasing the yield of biomass of plants, for example for paper and fuel production has previously been done by breeding programs, but in recent years there is interest in the use of genetic manipulation or plant modification for such purposes.
The division of cells to form the vascular tissue is a highly ordered process. Prominent polarity of cells destined to become either phloem cells or xylem cells is observed, the latter eventually forming the woody tissue of the plant. Xylem is principally water transporting tissue of the plant, and together with phloem, forms a vascular network for the plant. The cells of the xylem which are principally responsible for carrying water are the tracheary elements, of which there are two types—tracheids and vessels.
However, whilst there has been much investigation into the regulation of growth at the apical meristems, there is less understanding of the growth of the vascular tissue. Fisher et al (Current Biology 17 1061-1066 (2007)) report a loss of function mutant in which the spatial organisation of the vascular tissue is lost and the xylem and phloem cells are interspersed. The mutant is in a gene named PXY, which encodes a receptor-like kinase.
Tracheary elements (TEs) are cells in the xylem that are highly specialized for transporting water and solutes up the plant. They are produced from xylem cells by a process which involves specification, enlargement, patterned cell wall deposition, programmed cell death and cell wall removal. This results in adjacent TEs being joined together to form a continuous network for water transport.
Jun et al (supra) disclose that the CLE domain of CLE41 is identical to Tracheal Element Differentiation Inhibitory Factor (TDIF), which has been shown to inhibit cell differentiation, and CLE42 differs by only one amino acid from the TDIF sequence. When exogenously applied to cell cultures, synthetic CLE41 and CLE42 suppressed the formation of tracheary element cells from the xylem (Ito et al, supra).
There remains a need for identification of genetic elements, the manipulation of which can be used to alter the growth and/or structure of the plant.